Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection suppl (original) (raw)

Mouse embryonic stem cells with validated targeting of the Atg16l1 locus (exon 3 flanked by loxP sites) were purchased from EUCOMM (http://www.knockoutmouse.org/martsearch/project/23950). A reporter/resistance cassette composed of an FRT-flanked lacZ/neomycin sequence followed by a loxP site was placed upstream of the targeted exon. An additional loxP site was inserted downstream of the targeted exon. Exclusive splicing of the lacZ/neomycin cassette resulted in a reporter knockout mouse (Atg16l1 KO). After removal of the lacZ/neomycin cassette via Flpe-mediated recombination, the floxed Atg16l1 allele (Atg16l1 f/f) maintained wild-type-like expression until the critical exon was removed by Cre recombinase (Atg16l1 CKO). Microinjections of Atg16l1-targeted embryonic stem cells were performed at the Transgenic Core of Brigham and Women's Hospital (Boston, MA). To generate mice with conditional targeting of Atg16l1, heterozygotes of the targeted allele were bred with FLPe-expressing C57BL/6 mice (gift from Katia Georgopoulos, Massachusetts General Hospital, Boston, MA). Atg16l1 f/f mice were then bred with mice expressing cre recombinase under the control of villin (Villin-cre) or CD11c (CD11c-cre) promoters (gift from Joseph Avruch, Massachusetts General Hospital, Boston, MA; Villin-cre: Jax mice stock # 004586 and CD11c-cre: Jax mice stock #008068). Western Blotting Cell and tissue extracts were prepared using lysis buffer (100 mM Tris-HCl, 100 mM NaCl, 1% NP-40, 1mM DTT, 10mM NaF, 0.5mM Na 3 VO 4 , and one tablet Complete Mini protease inhibitor (Roche) per 10 ml lysis buffer; pH 8). Following SDS-PAGE (Bio-Rad), proteins were transferred onto PVDF 1 membranes (Immobilon-P, Millipore) and visualized using ATG16L1 antibody clone D6G5 (Cell Signaling Technology, 8089), p62 antibody (American Research Products, 03-GP62-C), actin antibody (Sigma Aldrich, A2066), and LC3B antibody (Sigma Aldrich, L7543); goat anti-rabbit HRP (Dako, P0448) [ATG16L1, actin, LC3B] or rabbit anti-guinea pig HRP (Dako, P0141) [p62] were used as secondary antibodies. Epithelial Cell Enrichment Briefly, terminal ileum, cecum, and colon were harvested from mice, sliced longitudinally to expose the luminal side of the tissues, and cut further into small pieces. After being washed in calcium-and magnesium-free PBS, tissues were incubated with 1 mM DTT in PBS, followed by 30 mM EDTA in PBS, all at room temperature. The enriched fractions were pelleted and stored at-20°C until lysis for Western blot. CD11c + Cell Isolation Single cell suspensions from spleen and MLN of mice (8-10 weeks of age) were obtained after digestion with Liberase TL (0.15 mg/ml, Roche) and DNase 1 (325 units/ml, Sigma-Aldrich) for 30 minutes at 37°C. Cell suspensions were enriched for CD11c + cells by depleting B220-, CD3-, Ter119-, and Gr1-expressing cells by incubating with biotinylated antibodies and anti-biotin magnetic beads. Labeled cells were removed from the cell suspension using magnetic MACS columns per the manufacturer's protocol (Miltenyi Biotec). Remaining non-labeled cells were incubated with 2.4G2 Fc block (BD Pharmingen) and stained with CD11c-PE antibody (BD Pharmingen). CD11c high cells were FACS-sorted using a MoFlo cell sorter; the purity of sorted cells was >99%. Cell pellets were stored at-20°C until lysis for Western blot.